Preparation and initial characterization ofbiodegradable particles containing gadolinium-DTPA contrast agent for enhanced MRIAmber L. Doirona, Kevin Chua, Adeel Alib, and Lisa Brannon-Peppasc,1

aDepartment of Biomedical Engineering, University of Texas, Austin, TX 78712; bLaw Offices of R. V. Reddy, 7171 Harwin Drive, Suite 203,Houston, TX 77036; and cAppian Labs, LLC, 11412 Bee Caves Road, Suite 300, Austin, TX 78738

Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved July 18, 2008 (received for review October 31, 2007)

Accurate imaging of atherosclerosis is a growing necessity fortimely treatment of the disease. Magnetic resonance imaging (MRI)is a promising technique for plaque imaging. The goal of this studywas to create polymeric particles of a small size with high loadingof diethylenetriaminepentaacetic acid gadolinium (III) (Gd-DTPA)and demonstrate their usefulness for MRI. A water-in-oil-in-oildouble emulsion solvent evaporation technique was used to en-capsulate the MRI agent in a poly(lactide-co-glycolide) (PLGA) orpolylactide-poly(ethylene glycol) (PLA-PEG) particle for the pur-pose of concentrating the agent at an imaging site. PLGA particleswith two separate average sizes of 1.83 �m and 920 nm, andPLA-PEG particles with a mean diameter of 952 nm were created.Loading of up to 30 wt % Gd-DTPA was achieved, and in vitrorelease occurred over 5 h. PLGA particles had highly negative zetapotentials, whereas the particles incorporating PEG had zeta po-tentials closer to neutral. Cytotoxicity of the particles on humanumbilical vein endothelial cells (HUVEC) was shown to be minimal.The ability of the polymeric contrast agent formulation to createcontrast was similar to that of Gd-DTPA alone. These resultsdemonstrate the possible utility of the contrast agent-loadedpolymeric particles for plaque detection with MRI.

atherosclerosis � imaging � targeted � PLGA � PEG

Despite recent advances in medicine and imaging, complica-tions arising from atherosclerosis remain the leading causes

of morbidity and mortality in the developed Western world withexpectations for growth in coming years (1). Additionally, thefinancial burden of cardiovascular disease is more than doublethat of all cancers and is estimated at $430 billion in the UnitedStates in direct and indirect costs for 2007 (1). There have beenmany advances in identifying arterial stenosis, but the mostcommon source of clinical events remains rupturing of vulner-able plaques in vessels with only 50–60% stenosis (2, 3). In fact,only 30–40% of cases of myocardial infarction have a plaque thatnarrows the artery enough to limit f low (3, 4). Luminal stenosisis delayed until the lesion occupies a significant area within thearterial lamina because of the phenomenon of vascular remod-eling (5). The need for stage-specific plaque imaging is para-mount because early detection is necessary to predict whichpatients require aggressive treatment for plaques vulnerable torupture (6).

The evolution of atherosclerotic plaque is complex andchronic in nature, spanning decades in development from thefirst microscopic evidence of lipid deposits in the arterial intimato occlusive or thrombus-forming complicated plaques. Athero-sclerotic plaques progress through distinct stages with molecularand cellular changes including endothelial dysfunction, the ac-cumulation of oxidized low-density lipoprotein and connectivetissue, the involvement of inflammatory and vascular cells,delineation of the plaque into lipid core and fibrous cap regions,and the appearance of fissures or other anomalies (7, 8). Specificstages during the lifespan of the plaque are more vulnerable torupture because of cellular, molecular, and macroscopic factors

including the size and composition of the lipid-rich core, thethickness and integrity of the fibrous cap, active inflammatoryresponses, and cap fatigue due to chronic repetitive mechanicalstresses (7–9). Because of the complex morphological and chem-ical changes involved in the cascade of atherosclerotic events,imaging of the plaque at a molecular or cellular level is of utmostimportance in predicting its likelihood for rupture and identi-fying the correct treatment.

As a noninvasive imaging modality, magnetic resonance im-aging (MRI) is very promising for atherosclerotic plaque imag-ing because it does not use ionizing radiation, provides high-resolution images of vasculature, is sequentially repeatable, andcharacterizes the composition of atherosclerotic plaques (10–12). MRI can discriminate plaque constituents such as the lipidcore and fibrous cap, plaque burden, and vessel stenosis on thebasis of biochemical and biophysical factors (13). Several ap-proaches such as cardiac and respiratory gating, new scansequences, and the use of contrast agents are being used toimprove the spatial resolution and sensitivity limitations of MRIas is necessary for imaging of coronary atherosclerotic plaques.

Drug delivery has long been researched to improve therapeu-tic efficacy while reducing side effects, and many of the sameprinciples used in this field also augment the delivery of imagingagents to specific physiological locations. Poly(lactide-co-glycolide) is a widely used polymer for these drug deliverysystems because it is biodegradable, biocompatible, and widelyavailable, and has been FDA-approved for many biomedicalapplications (14). Targeted delivery utilizes tissue- or cell-specific phenotypic characteristics to concentrate an active agentat a desired location. Targeted delivery has not only been usedfor therapeutic purposes but has also recently been researchedfor its ability to concentrate imaging or contrast agents at alocation for the detection of malignancies and for monitoring theeffects of therapeutic agents (15).

Gadolinium diethylenetriaminepentaacetic acid is a positiveMR contrast agent. This agent has a long history in MR, has beenFDA-approved for nearly two decades, and is a well character-ized stable chelate. Directly labeling a gadolinium chelate withantibody for targeted delivery has not provided sufficient con-centration of agent at the targeted site for in vivo imaging, sodelivery of a large payload of agent to the site of interest is

This paper results from the Arthur M. Sackler Colloquium of the National Academy ofSciences, “Nanomaterials in Biology and Medicine: Promises and Perils,” held April 10–11,2007, at the National Academy of Sciences in Washington, DC. The complete program andaudio files of most presentations are available on the NAS web site at www.nasonline.org/nanoprobes.

Author contributions: A.L.D. and L.B.-P. designed research; A.L.D., K.C., and A.A. performedresearch; A.L.D., K.C., A.A., and L.B.-P. analyzed data; and A.L.D. and L.B.-P. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: lpeppas@etibio.com.

© 2008 by The National Academy of Sciences of the USA

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imperative (16, 17). Faranesh and colleagues (18) demonstratedthe utility of the inclusion of small amounts of Gd-DTPA inPLGA microparticles for tracking a drug delivery vehicle. Ad-ditionally, several groups have created gadolinium-containingmicelles, microspheres, nanoparticles, and liposomes for thepurposes of targeting delivery or modifying the biodistribution toincrease the site-specific concentration of gadolinium over thatof aqueous gadolinium or gadolinium directly conjugated to thetargeting molecule (19–26). Toward the same goals, this articledetails a method for the entrapment of hydrophilic agents,specifically Gd-DTPA, with very high loading in a polymericparticulate system as well as work toward achieving a targetableimaging agent particle formulation for the detection of stagedatherosclerosis.

ResultsAnalysis of PLGA Particles. Particles of two different size distribu-tions were synthesized by using a water-in-oil-in-oil (W/O/O)double emulsion method, as schematically shown in Fig. 1, using

sonication at either 45 or 120 W during the formation of theW/O/O emulsion. The two particle types were characterizedseparately on the basis of morphology, size, zeta potential, andGd-DTPA loading. Additionally, the release profile of Gd-DTPA from the particles was determined, and the cytotoxicityof the larger Gd-DTPA-loaded PLGA particles was found byusing HUVEC to mimic the arterial endothelium.

Analysis of PLA-PEG Particles. PLA-PEG was polymerized by usingthe ring opening solution polymerization of lactide onto CM-PEG. The structure of the polymer was confirmed by using1H-NMR, and the number average molecular weight was foundby using gel permeation chromatography (GPC) to be �55 kDawith a polydispersity (Mw/Mn) near 1.08, depending on the batch.PLA-PEG nanoparticles were produced by using the W/O/Omethod with sonication at a 45-W sonic output power, and theseparticles were evaluated on the basis of morphology, size, zetapotential, and Gd-DTPA encapsulation efficiency and loading.

Particle Characterization. The W/O/O method consistently pro-duced near-spherical particles with smooth surfaces, little ag-glomeration, and a general size range of 20 nm to 20 �m. Fig. 2shows the surface characteristics of each particle type as exam-ined by scanning electron microscopy (SEM), and Fig. 3 showssome of the smaller particles as studied with transmission

Fig. 1. Schematic of W/O/O protocol showing use of sonication (45 W) orprobe sonication (120 W) during formation of second emulsion.

Fig. 2. Scanning electron micrograph of particles containing Gd-DTPA prepared by using the W/O/O emulsion technique. (a) PLGA particles prepared by using45-W sonication for both emulsions. (b) PLGA particles prepared by using 120-W sonication during formation of W/O/O emulsion. (c) PLA-PEG particles.

Fig. 3. Transmission electron micrograph of negatively stained PLGA parti-cles prepared by using the W/O/O emulsion with 120-W sonication.

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electron microscopy (TEM). Fig. 2 highlights the differences inparticle sizes and size distributions resulting from the differentformulation methods. The PLGA particles (Fig. 2 a and b) allhad a rather large size distribution, whereas the PLA-PEGparticles (Fig. 2c) had a much narrower size distribution.

Zeta Potential and Sizing. Table 1 shows the size ranges andpolydispersity indices for each of the three described particletypes. The PLGA particles formed by using 45-W sonication forboth emulsions were larger than the PLGA particles formed withthe probe sonicator at a 120-W output power. The sizes of PLGAparticles with the larger mean diameter were measured with aCoulter Nanosizer, whereas the size distributions of the smallerPLGA particles and PLA-PEG particles were measured by usinga ZetaPlus instrument. The average zeta potentials of theunloaded PLGA particles differed little with the change in size;however, the PLA-PEG particles had an average zeta potentialmuch closer to neutral. The Tyndall effect was seen for eachbatch as the particles resuspended well after freeze-drying.

Encapsulation and Loading. Encapsulation and loading as mea-sured by inductively coupled plasma mass spectrometry (ICP-MS) varied depending on the targeted loading of Gd-DTPA, butencapsulation efficiency within PLGA particles (1.83 �m) wastypically 30–70% with this method with a maximum Gd-DTPAloading over 50 wt %. The smaller PLGA particles (920 nm) hadtypical encapsulation efficiencies of 20–50% and loadings up to25 wt % Gd-DTPA. PLA-PEG particles had encapsulationefficiencies up to 70% and loading up to 15 wt %.

In Vitro Release. The in vitro release profile of Gd-DTPA fromPLGA particles (1.83 �m) at various loadings in PBS at 37°C isshown in Fig. 4. The release occurred quickly with �90%released in the first hour because of the high hydrophilicity of theentrapped agent. The release was monophasic and occurred over5 h. The release from smaller PLGA particles (920 nm) followsthe same profile and occurs in the same time period, as shownin Fig. 5.

Cytotoxicity. The effects on the viability of human umbilical veinendothelial cells in vitro after exposure for 24 h to microparticles(1.83-�m average diameter, targeted loading 20 wt % Gd-DTPA) at concentrations of 10, 20, 50, 100, 200, 500, and 1,000�g/ml were determined by using an MTS assay, and results areshown in Fig. 6. Additionally, Fig. 7 shows the cumulative effecton HUVEC of tumor necrosis factor � (TNF-�) and PLGAparticles (1.83 �m, targeted loading 20 wt % Gd-DTPA) at thesame concentrations as above. The microparticles only margin-ally affected cell viability at high concentrations, and no resultswere statistically significant from the control as confirmed byANOVA.

MRI of Particles. The PLGA particles (1.83 �m) loaded withGd-DTPA effected a change in contrast on MR images ascharacterized by their longitudinal relaxivity (r1) as evaluated byusing a 1.5-T scanner. Increased loading brought about anincrease in r1 values, but the Gd-DTPA-loaded microparticleshad little effect on r2 values, as was expected at these concen-trations. The particle-specific longitudinal relaxivity was foundto be 2.37 ml�s�1�mg�1 for particles with 54.5% Gd-DTPAloading. The relaxivity of the particle formulation was shown tobe similar to that of unencapsulated Gd-DTPA.

DiscussionThe PLGA and PLA-PEG particles formulated with the W/O/Omethod are suitable for encapsulation of Gd-DTPA for thepurpose of creating a targetable MR contrast agent for thedetection of atherosclerosis. The application of a double emul-sion solvent evaporation formulation technique with a hydro-phobic oil continuous phase to create small microparticles andnanoparticles to entrap Gd-DTPA has been described. Theparticles are spherically shaped, stable, and not agglomerated.The encapsulation of Gd-DTPA with this W/O/O protocol issimilar to or higher than that shown with other encapsulationmethods (18–27). Most importantly, the Gd-DTPA-loaded mi-croparticles were shown to augment contrast in in vitro studiessimilarly to Gd-DTPA alone. The entrapment of Gd-DTPA ina particle allows for the possibility of achieving a high concen-tration of contrast agent at the imaging site as well as targeting.

Release of Gd-DTPA occurs over a time period appropriatefor clinical imaging. The Gd-DTPA is released quickly becauseof the propensity of the agent to partition into aqueous media.The imaging agent is entrapped within the biodegradable PLGAcore and is released through a combination of diffusion throughthe polymeric matrix and the degradation of the matrix itself.Future projects may aim to contain release more completelyduring an imaging time period before allowing Gd-DTPA escapefrom particles. However, a balance must be found between

Table 1. Particle characteristics

Particle typeAverage

size PolydispersityZeta potential,

mV

PLGA (45-W sonication) 1.83 �m 6 � 2 �51 � 8PLGA (120-W sonication) 920 nm 0.16 �42 � 6PLA-PEG (45-W sonication) 952 nm 0.14 �9 � 3

PLGA (45-W sonication) size was measured by using the Coulter Nanosizer;PLGA (120-W sonication) and PLA-PEG particle sizes were measured with theZetaPlus instrument.

Fig. 4. Release of Gd-DTPA from PLGA particles (1.83 �m) at various load-ings. Error bars indicate standard deviation of triplicate samples at a givenpoint.

Fig. 5. Release of Gd-DTPA from PLGA particles (920 nm) at various loadings.Error bars indicate standard deviation of triplicate samples at a given timepoint.

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allowing water into the polymer matrix and preventing Gd-DTPA release because the central Gd3� moiety must be hy-drated to augment contrast.

Minimal cytotoxic effects were seen even when HUVEC wereexposed to high concentrations of particles. This might mimic thesituation where targeting concentrates many particles at onelocal site. The HUVEC line mimics the arterial endotheliumduring early development of atherosclerosis and was activated inthis study by using TNF-� for the purpose of examining the effectof PLGA particles on HUVEC with up-regulated cell-surfacereceptors such as cell-adhesion molecules.

The size range of particles produced by using the W/O/Oprotocol can be adjusted depending on the type of sonicationused. The use of a probe sonicator has decreased the particlesize considerably and reduced the presence of microparticlesin the formulations. The two differently sized PLGA particleformulations have been shown here to share similar encapsu-lation capabilities and release profiles of Gd-DTPA. Theadvantage of having more than one size distribution is high-lighted by the need for a particle that reaches the plaquesurface in vivo. Particle size has been shown to theoreticallyaffect margination speed and the time needed to reach thevessel wall by circulating particles (28). The complex f luiddynamics in the area of plaque development may necessitateslightly larger particles to overcome being swept away andnever entering the boundary layer near the plaque.

As is well known, a particulate formulation has a smalllikelihood of reaching its desired target before opsonization andrecognition by the reticuloendothelial system (RES) unless amethod is in place to block this process. Poly(ethylene glycol)(PEG) is widely used to accomplish this goal of reducingopsonization and increasing circulation time from seconds orminutes up to hours (29). This laboratory has investigated the useof a poly(lactic acid)-poly(ethylene glycol) block copolymer withthe W/O/O formulation method to synthesize particles with a

higher likelihood to bypass the RES. Additionally, the size oftheses particles is reduced compared to PLGA particles possiblybecause of the presence of both hydrophilic and hydrophobicregions in the PLA-PEG polymer. The mean diameter of thePLA-PEG particles was 952 nm with a polydispersity of 0.14.Additionally, zeta potential values closer to neutral than those ofPLGA particles reflect changes in surface charge that transpirewith the incorporation of PEG.

In addition to the work shown here, this W/O/O method hasbeen used by our laboratory for the encapsulation of other activeagents such as Rhodamine 6G. High encapsulation of Rhoda-mine 6G has been achieved with a slow release occurring overseveral weeks. This formulation is useful in cellular studies totrack particles and their cellular interactions using fluorescence.The possibility for coencapsulation of two imaging agents inthese particles exists and is attractive for the study of the particlesby using more than one imaging modality.

Materials and MethodsSynthesis of PLGA Particles. Particles were synthesized by using a water-in-oil-in-oil (W/O/O) double emulsion solvent evaporation method based on a proce-dure described by Chaw and colleagues (30). Several steps in the protocol wereoptimized for particle morphology and size as well as entrapment of the activeagent Gd-DTPA (data not shown). These optimized factors included emulsifierconcentration, type of mechanical agitation (sonication, vortexing, stirring, etc.),centrifugation speed and time, and inclusion of cryoprotectant during freezingand freeze-drying to arrive at this final protocol. The PLGA polymer (Medisorb;Mn � 11 kDa) was dissolved at a concentration of 80 mg/ml in 1.25 ml of acetone(HPLC grade; Fisher Scientific) to form the polymer phase. Diethylenetriamine-pentaacetic acid gadolinium (III) dihydrogen salt hydrate (Sigma) was dissolved inan aqueous solution of BSA (10 mg/ml; Sigma) at a concentration of 0–50 mg/ml.The external oil phase was comprised of egg lecithin (Acros) at a concentration of0.0625 mg/ml in light mineral oil (Sigma). The aqueous phase (500 �l) was addedto the polymer phase and sonicated at an average sonic power of 45 W (VWRModel 50D). This primary W/O emulsion was subsequently added to the externaloilphase(25ml)andeithersonicatedwithabench-topsonicatorat45Wfor1min

Fig. 6. Viability of HUVEC when exposed to particles (1.83 �m, targeted loading of 20 wt % Gd-DTPA) at concentrations shown in legend as compared to control.

Fig. 7. Viability of HUVEC activated by TNF-� when exposed to particles (1.83 �m, targeted loading of 20 wt % Gd-DTPA) at concentrations shown in legendas compared to control.

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to create the larger average size particles or for 10 min with a Misonix XL2020probe sonicator at an output power of 120 W to create the smaller sized particles.The solution was stirred at 500 rpm during vacuum evaporation for 45 min toremove organic solvent. Particles were collected with centrifugation at 43,000 �g for 15 min at 4°C and washed three times with hexane (HPLC grade; Acros) toremovemineraloil.Hexanewasremoved,andtheparticleswerefrozenfor12–24h and freeze-dried (Labconco Freeze Dryer 4.5) for a period of 48 h. Particles werestored at �20°C until further analysis.

Synthesis of PLA-PEG Particles. Poly(lactic acid)-poly(ethylene glycol) block co-polymer was prepared for use with the W/O/O particle formulation method. ThePLA-PEG copolymer was prepared by ring opening polymerization of lactide inthe presence of carboxymethyl-poly(ethylene glycol) (CM-PEG), using a proce-dure modified from Quellec and colleagues (31). Briefly, recrystallized DL-lactidemonomer (Polysciences) was polymerized onto CM-PEG (molecular weight 3,400;Laysan Bio) in the presence of stannous 2-ethylhexanoate (Sigma) in toluene(anhydrous; Acros) at 110°C under a nitrogen atmosphere. The product wasrecovered by centrifugation after dissolution in methylene chloride (Fisher Sci-entific)andprecipitation inethylether (anhydrous;FisherScientific).Thepolymerproduct was confirmed by 1H-NMR, and the molecular weight was quantified byusing GPC. The PLA-PEG was then used to synthesize particles by the W/O/Omethod in the same manner described above, using the bench-top sonicator forboth sonication steps. The addition of trehalose as a cryoprotectant duringfreezingandfreezedryingaidedinstabilizingparticlesagainstagglomeration,assuggested by other investigators (32, 33).

Particle Characterization. Particles were analyzed by using SEM for morphologyand size. Particles were evaluated on the basis of shape, surface smoothness, andthe presence of agglomeration or interparticular bridging. Additionally, the sizerange was judged qualitatively by SEM. Particle samples for SEM were preparedby suspending a small amount of the lyophilized particles in deionized water andallowingthesuspensiontodryonconductivetapeontopofametallic studbeforesputter coating with gold. Images were captured at a 10-kV accelerating voltageon a Hitachi S-4500 field emission SEM. A Phillips EM 208 transmission electronmicroscope (TEM) was used to confirm particle size and to study samples at highmagnification. Particles were suspended in deionized water, dried on a 300 meshcopper TEM grid with a carbon film (Electron Microscopy Sciences), and stainedwith uranyl acetate (Baker Chemical).

Zeta Potential and Sizing. Zeta-potential measurements were taken to verify thestability of the colloid suspension. Gd-DTPA interfered with zeta-potential mea-surements as it increased the conductance of the solution, so tests were run withparticles formulated by the W/O/O method in the absence of Gd-DTPA. Lyophi-lized particles were suspended at 1 mg/ml by sonication in a 1 mM KCl electrolytesolution, and the zeta potential and size were measured at 25°C with a ZetaPlusinstrument with accompanying software (Brookhaven Instruments). The instru-ment used electrophoretic light scattering and the laser Doppler velocimetrymethod to measure the zeta potential and number average distribution ofparticle sizes. The size distributions of the PLGA particles formulated by using theprobe sonicator and the PLA-PEG particles were measured by using the ZetaPlus,and the polydispersity was given as a reading from 0 to 1. The size of PLGAparticles formulated by using the bench-top sonicator for both sonication stepswas measured with a Coulter Nanosizer dynamic light scattering instrument withthe polydispersity given as an index from 0 to 9. As mentioned above, the size wasalso qualitatively verified by using SEM, and all measurements were done onpreviously lyophilized samples.

Encapsulation and Loading. Active agent loading and encapsulation efficiencywere determined by quantifying the amount of Gd in a representative sample ofeach Gd-DTPA particle batch by ICP-MS, using an OptiMass 8000 spectrometer

with OptiMass software (version 1.2; GBC Scientific Equipment). The correctisotopic ratios were confirmed, and the absence of interference by the polymerwas verified. A known mass of particles was suspended in deionized distilledwater by sonication, and volumetric dilutions were done to achieve an approx-imate Gd concentration within the detection limits of the spectrometer. Countsof Gd were correlated to a concentration of Gd-DTPA based on a standard curvegenerated by using Gd-DTPA.

In Vitro Release. A known mass of lyophilized particles was suspended in 20ml of PBS (Sigma) at pH 7.4 and left in a 37°C waterbath for a specifiedperiod. Samples were centrifuged at 43,000 � g for 15 min at 20°C, and 75%of the supernatant was collected and replaced with fresh PBS. Supernatantsamples were analyzed by ICP-MS as described above. This procedure wasrepeated until no additional release was detected, and the remainingpellet was analyzed for unreleased Gd-DTPA. Release assessed by this statictest did not to differ significantly from release occurring with sampleagitation, and thus the static test was used because of simplicity.

Cytotoxicity. The cytotoxicity of the Gd-DTPA-loaded PLGA particles was deter-minedbyusingacolorimetric3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay based on the conversion of thetetrazolium salt into a formazan product by viable cells. The characteristic absor-bance of the formazan product was used to determine the percentage of viablecells when compared to control cells grown without exposure to particles. TNF-�(Sigma) was used to up-regulate cell-adhesion molecules on HUVEC to be usedlater fora simple invitro targetingscheme.PooledHUVEC(Lonza/Clonetics)werecultured for 24 h in endothelial growth medium (EGM) (Lonza/Clonetics) at 37°Cwith 5% CO2 in a 96-well plate after splitting from T-25s at second passage (2,000cells per well). Plate wells and flasks were coated with 0.1% gelatin (Difco/BectonDickinson) in Dulbecco’s PBS (Mediatech) 3 h before plating with cells. Cells wereexposed to Gd-DTPA-loaded PLGA particles (targeted loading 20 wt % Gd-DTPA,45-W sonication) for 24 h and 0 or 10 ng/ml TNF-� for 8 h. Promega CellTiter 96AQueous One Solution Cell Proliferation Assay was added to the wells andallowed to incubate for 4 h. The absorbance was measured at 490 nm and at areference wavelength of 700 nm to assess viability of cells in the presence of PLGAparticles compared to cells not exposed to particles. A one-way ANOVA was runto examine statistical differences in absorbance after verifying that the datafollowed a normal distribution.

MRI of Particles. In vitro r1 and r2 relaxivities were determined for Gd-DTPA-loaded PLGA particles and unloaded PLGA particles suspended at 1 mg/ml inagarose gels (1.5 wt % agarose prepared with deionized water), using a 1.5-Tscanner (Excite HD; GE Healthcare) accompanied by a GE Quad Ankle coil.Suspended particles containing Gd-DTPA from 0 to 0.5 mM were scanned andcompared to the same concentrations of unencapsulated Gd-DTPA. For T1

measurements, an inversion recovery (2D IR) sequence was used with thefollowing imaging parameters: TR � 3,000 ms; TE � 14 ms; TI � 50, 70, 100, 200,250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 2,950 ms; band width of15.6 kHz; FOV of 18 � 9 cm; slice thickness of 4.0 mm; matrix of 256 � 128; anda flip angle of 90°. For the T2 measurements, a 2D SE sequence was used witha TR � 4,000 ms; TE � 16, 20, 30, 40, 60, 80, 100, 200, 300 ms; band width of15.6 kHz; FOV of 18 � 9 cm; slice thickness of 4.0 mm; matrix of 256 � 128; andflip angle of 90°. Values for r1 and r2 were determined where r1 and r2 are theinverse of T1 and T2, respectively.

ACKNOWLEDGMENTS. We thank John Hazle, Ph.D., and Andrew Elliot, Ph.D.,for their help with magnetic resonance imaging at M. D. Anderson CancerCenter, as well as James Holcolmbe, Ph.D., Thomas Kreschollek, Ph.D., andHaley Finley-Jones for the ICP-MS work. This work was supported by theAmerican Heart Association and the National Science Foundation IntegrativeGraduate Education and Research Traineeship (IGERT) program.

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